1. Field of the Invention
This invention relates to a method and a kit for the rapid detection of pathogenic organisms in environmental samples using the polymerase chain reaction. More specifically, it relates to methods for differentiating among various species of pathogenic and non-pathogenic Yersinia bacteria by specific restriction enzymes in conjunction with the polymerase chain reaction.
2. Description of the Prior Art
Bacterial contamination of food, water, or soil can result in serious illness in humans and animals. Detection and identification of pathogenic organisms are important for monitoring unwarranted biological attacks, for containment of potential epidemics, for elimination of natural host reservoirs, for prevention of further contamination, and for appropriate subsequent treatment should exposure occur. Rapid detection and identification of the sources of contamination provides the information to properly eliminate and prevent the spread of bacteria so as to ensure the quality and safety of food and water resources, and the prevention of epidemics or biological attacks. The polymerase chain reaction, herein referred to as PCR, is an in-vitro method of amplifying DNA sequences. Target DNA from bacterial, viral, fungal, parasitic or biological source of interest, may be amplified and detected in minute quantities, theoretically as small as one molecule. Practical applications have used 100 organisms, or the equivalent of 10 nanograms of DNA, for successful identification. The target DNA to be amplified must be flanked by a known sequence of several nucleotides; short pieces of DNA are synthesized with sequences identical to the known sequences; these are referred to as oligonucleotide primers.
At present, there is no known methodology for identifying Yersinia bacterial species using restriction enzymes in conjunction with PCR. There is a need, therefore, for a rapid and convenient method for identifying such bacteria.
The PCR process in accordance with this invention requires denaturing the target DNA strand into two separate strands by heating it to 93-96 degrees C., preferably 95 degrees C. A predetermined primer is then annealed to each of the separate strands at the flanking positions at 37-60 degrees C., preferably 55 degrees C. A heat-resistant enzyme, referred to as Taq polymerase, synthesizes a strand of DNA complementary to the existing DNA strands to form two complete double DNA copies of the original starting target DNA. By repeating this process once, four double-stranded chains are formed. Every time the process is repeated, the amount of PCR product is exponentially increased. The conditions of the PCR process may be slightly different, depending on the primers and DNA template used.
Electrophoresis is used to display patterns that differentiate between the DNAs of different genera of the bacteria in the sample. A restriction enzyme is added to the PCR product to generate an electrophoresis pattern specific to the individual species of bacteria.